The invention resides in the technical fields of virology and molecular genetics.
Papillomaviruses are small, nonenveloped, icosahedral DNA viruses that replicate in the nucleus of squamous epithelial cells. Papillomaviruses consist of a single molecule of double-stranded circular DNA about 8,000 base pairs (bp) in size within a spherical protein coat of 72 capsomeres. Such papillomaviruses are classified by the species they infect (e.g., bovine, human, rabbit) and by type within species. Over 50 distinct human papillomaviruses (xe2x80x9cHPVxe2x80x9d) have been described. See, e.g., Fields Virology (3rd ed., eds. Fields et al., Lippincott-Raven, Philadelphia, 1996).
The DNA of many papillomaviruses, including over 50 human viruses, has been cloned and sequenced. Although there is a high degree of sequence divergence between species, all papillomaviruses share some common features of genome organization. That is, the genome is subdivided into an early region containing proteins E1-E8 (not all are present in all species), a late region, containing genes L1 and L2, and a long control region (LCR) of transcription, including the promoter and enhancer for the viral early genes and the origin of replication. The early region encodes genes required for viral DNA replication, cellular proliferation, and, in some viruses, cellular transformation. The E1 gene and E2 gene encode E1 and E2 polypeptides that bind to the LCR region and induce replication from the origin of replication in the LCR region. The E5, E6 and E7 proteins of the different papillomaviruses have proliferation and sometimes transforming activities. The late region (about 3 kb) codes for the capsid proteins. L1 is the major capsid protein and is relatively well conserved among all the papillomavirus types. The L1 proteins is about 500 amino acids in size. L1 probably induces the major humoral and cell-mediated responses to viral infection. The L2 proteins are about 500 amino acids in size, account for only a small proportion of the virion mass, and their function is not yet clear. The LCR region contains an origin of replication with binding sites for E1 and E2 and other cis acting sequences in the promoter and enhancer region.
Papillomaviruses display a marked degree of cellular tropism for epithelial cells. Specific viral types have a preference for either cutaneous or mucosal epithelial cells. All papillomaviruses have the capacity to induce cellular proliferation. The most common clinical manifestation of proliferation is the production of benign warts. However, many papillomaviruses have capacity to be oncogenic in some individuals and some papillomaviruses are highly oncogenic.
None of the papillomaviruses can be propagated in monolayer cell culture to yield virUs particles, probably because full epithelial differentiation required for production of infectious viral particles is not achieved in conventional cell cultures. However, bovine papillomavirus (BPV) can undergo stable episomal replication in several transformed cells including transformed fibroblasts. Certain anogenital HPVs can grow in organogenic (raft) cultures of epithelial cells (Meyers et al., Science 257, 971-973 (1992) and Dollard, et al., Genes Dev. 6, 1131-1142 (1992)) but this system requires that one start with cells that harbor already episomal HPV genomes. At present only two such cell lines have been described: the CIN612 line, which harbors highly oncogenic anogenital HPV-31 genomes (Bedell et al, J. Virol., 65, 2254-2260 (1991) and the W12 cell line, which harbors episomal highly oncogenic anogenital HPV-16 genomes (Sterling et al., J. Virol. 64, 6305-6307 (1990). Stable replication in cell culture has not been shown so far for any benign cutaneous HPV. Because of the relative difficulties of propagation, most studies of viral lifecycle and protein function have been done in transformed fibroblasts with BPV.
Several authors have discussed papillomaviruses as possible vectors for gene therapy. Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al., WO 94/12629 and U.S. Pat. No. 5,674,703 and Xiao and Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996). The attraction of papillomaviruses for this role arises from their persistent but nonfatal state of infection in the human body, their capacity for episomal replication, and their specificity for epithelial cells, which constitute an ideal target for many applications of gene therapy. The challenge in constructing papillomavirus based vectors is to incorporate a coding sequence for a therapeutic product without forfeiting or impairing viral functions that would allow long-term retention and expression of the therapeutic sequence in epithelial cells.
Woo et al., supra, report attempts to construct a bovine papillomavirus (BPV) vector for gene therapy. In a first experiment they reported that a vector containing a marker gene, together with BPV E1 and E2 sequences linked to endogenous BPV promoters, and LCR sequences was unable to replicate in a variety of cell types. However, when the endogenous promoters were replaced with a CMV promoter stable episomal replication was obtained. The authors conclude that the minimal elements for a BPV vector are E1 and E2 coding sequences, a foreign promoter, a BPV origin of replication and a vector maintenance sequence described by Lusky et al., Cell 36, 391-401 (1984). The authors speculate that the same approach could be used to produce a human papillomavirus but do not taken into account the different properties and replication capacities of human and bovine viruses. The authors also fail to take into account the lack of sequence similarity between human and bovine papillomaviruses and in particular, the lack of an equivalent to the bovine maintenance sequence of Lusky et al. in human papillomaviruses. Subsequently, it has been reported that the maintenance sequence of Lusky et al. does not appear to contribute to episomal stability even in BPV vectors. Ustav et al., EMBO 15, 1-11 (1996).
Ohe et al., Human Gene Therapy 6, 325-333 (1995) have reported a bovine papillomavirus vector in which early open reading frames E5, E6 and E7 were deleted. The vector was reported to undergo multicopy episomal replication in a variety of transformed or semitransformed cell types, and to support expression of an exogenous human gene. However, this type of vector is not ideal for gene therapy because it is possible that residual oncogenic activity residues in one of the remaining bovine papillomavirus proteins, and because E1 and E2 proteins are known to be immunogenic (Frattini et al., EMBO J. 16, 318-331 (1997)).
Ustav et al., WO 97/24451 also discuss production of a BPV vector for gene therapy. Ustav et al. report that BPV-1 contains 17 E2 binding sites within the LCR region having variable affinity. Ustav et al. propose that a key feature of a BPV vector is a maintenance element including multiple BPV E2 binding sites, of which E2 sites numbered 6, 7 and 8 appear particularly important. However, human papillomaviruses typically have only four E2 binding sites in the LCR element and have no equivalent sequences to BPV E2 binding sites 6, 7 and 8.
The present invention provides vectors derived from a human papillomavirus suitable for expressing a foreign gene in gene therapy.
The invention provides human papillomavirus vectors useful in gene therapy. Such a vector contains E1 and E2 coding regions from a benign or low risk human papillomavirus operably linked to a promoter and enhancer, and an LCR region from a human papillomavirus comprising an origin of replication including binding sites for the E1 and E2 proteins. The vector also contains a protein coding sequence operably linked to a second promoter and enhancer. In some vectors, the E1 and E2 coding regions are operably linked to their natural promoter and enhancer. In some vectors, the E1 and E2 proteins are from HPV-2, HPV-27 or HPV-57. In some vectors, the E1 and E2 coding regions, the promoter and enhancer, and the LCR region are present in a contiguous segment from HPV-2, -27 or -57 or chimeras thereof. Such cells can be designed to be capable of sustained expression of the protein coding sequence and/or episomal replication in epithelial vectors.
The invention further provides methods for generating and selection of new vector variants with improved properties by DNA shuffling.
The invention further provides methods of gene therapy. The methods entail introducing a vector, as described above, into the skin of a patient. The vector is expressed in cutaneous epidermal cells of the patient to produce the protein. In such methods, the vector is preferably expressed in cutaneous epidermal cells of the patient for at least two weeks. In some methods, the vector is introduced into the patient in naked form or encapsulated in liposomes. The protein to be expressed from such vector may, for example, serve to compensate for a defective human gene, induce a protective immunogenic response. In some methods, the protein to be expressed from the vector is linked to an inducible promoter. In such methods, expression can be controlled by treating a patient with a drug that induces expression. Optionally, the drug can be administered via a patch.
The invention further provides methods of producing an agent for promotion of hair growth or alteration of hair color. Such methods start by shuffling a population of variant forms of a gene encoding a potential agent to produce recombinant forms of the gene. A vector comprising E1 and E2 open reading frames from a cutaneous human papillomavirus (HPV) operably linked to a promoter and enhancer, and an LCR region including an origin of replication, and the recombinant sequences operably linked to a promoter and enhancer is then introduced into human skin present in a human or a nonhuman animal grafted with the human skin. The recombinant sequences are expressed, and expression product(s) thereof with hair-growth promoting activity stimulate growth of hair from the follicles. Vector is recovered from follicles showing hair growth or altered color. The steps are then repeated in an iterative fashion with recombinant sequences from the recovered vector forming the substrates for shuffling until an agent with desired activity of promoting hair growth or altering color has been identified.